Many animal behaviors, including feeding and reproduction, are controlled by neurons that release neuropeptides. The occurrence and frequency of such behaviors is determined by the firing pattern of such neurons. This project will investigate the mechanisms that regulate the excitability of the bag cell neurons of Aplysia, a group of neurons that controls reproductive behaviors lasting several hours. In response to brief synaptic stimulation, these neurons generate about 30 minute after-discharge that triggers the release of neuropeptides. This is followed by a prolonged period of inhibition, termed the refractory state, which persists for about 18 hrs and limits the occurrence of the behaviors. We plan to determine the cellular mechanisms that produce the refractory state, which is associated with a prolonged enhancement of potassium currents. The contribution of two splice variants of the SIo gene, which encode calcium-activated potassium channels, to the refractory state will be determined using native neurons, heterologous expression and RNA interference approaches. Immunochemical experiments will establish whether the SIo channels are coupled to protein kinase C-regulated calcium channel subunits, which are localized to neuropeptide release site and may be modulated by insertion and removal from the plasma membrane. Secondly, we plan to test the hypothesis that recruitment of a Kv2-family potassium channel to ring-shaped clusters on the plasma membrane also contributes to the onset of refractoriness. Finally, we plan to investigate a protein kinase C-regulated, non-selective cation channel that provides the depolarization for afterdischarge. By molecular cloning and genetic manipulations, we plan to test the hypothesis that this channel is a member of the pkd and/or trp family of cation channels. We shall also determine whether the physical association of the channel with protein kinase C becomes altered during the refractory state. An understanding of how ion channels are regulated in peptidergic neurons may lead to an understanding of normal and pathological changes in behavior, and provide insights into pathological changes of excitability such as epileptic seizures.